Mapping allosteric communications within individual proteins.

Allostery in proteins influences various biological processes such as regulation of gene transcription and activities of enzymes and cell signaling. Computational approaches for analysis of allosteric coupling provide inexpensive opportunities to predict mutations and to design small-molecule agents to control protein function and cellular activity. We develop a computationally efficient network-based method, Ohm, to identify and characterize allosteric communication networks within proteins. Unlike previously developed simulation-based approaches, Ohm relies solely on the structure of the protein of interest. We use Ohm to map allosteric networks in a dataset composed of 20 proteins experimentally identified to be allosterically regulated. Further, the Ohm allostery prediction for the protein CheY correlates well with NMR CHESCA studies. Our webserver, Ohm.dokhlab.org, automatically determines allosteric network architecture and identifies critical coupled residues within this network.

An intramembrane sensory circuit monitors sortase A-mediated processing of streptococcal adhesins.

Bacterial adhesins mediate adhesion to substrates and biofilm formation. Adhesins of the LPXTG family are posttranslationally processed by the cell membrane-localized peptidase sortase A, which cleaves the LPXTG motif. This generates a short C-terminal peptide (C-pep) that remains in the cell membrane, whereas the mature adhesin is incorporated into the cell wall. Genes encoding adhesins of the oral bacterium Streptococcus gordonii were differentially expressed depending on whether the bacteria were isolated from saliva or dental plaque and appeared to be coordinately regulated. Deletion of sspA and sspB (sspAB), both of which encode LPXTG-containing adhesins, unexpectedly enhanced adhesion and biofilm formation. C-peps produced from a model LPXTG-containing adhesin localized to the cell membrane and bound to and inhibited the intramembrane sensor histidine kinase SGO_1180, thus preventing activation of the cognate response regulator SGO_1181. The absence of SspAB C-peps induced the expression of the scaCBA operon encoding the lipoprotein adhesin ScaA, which was sufficient to preserve and even enhance biofilm formation. This C-pep-driven regulatory circuit also exists in pathogenic streptococci and is likely conserved among Gram-positive bacteria. This quality control mechanism ensures that the bacteria can form biofilms under diverse environmental conditions and may play a role in optimizing adhesion and biofilm formation.

Synchronous opening and closing motions are essential for cAMP-dependent protein kinase A signaling.

Conformational fluctuations play a central role in enzymatic catalysis. However, it is not clear how the rates and the coordination of the motions affect the different catalytic steps. Here, we used NMR spectroscopy to analyze the conformational fluctuations of the catalytic subunit of the cAMP-dependent protein kinase (PKA-C), a ubiquitous enzyme involved in a myriad of cell signaling events. We found that the wild-type enzyme undergoes synchronous motions involving several structural elements located in the small lobe of the kinase, which is responsible for nucleotide binding and release. In contrast, a mutation (Y204A) located far from the active site desynchronizes the opening and closing of the active cleft without changing the enzyme's structure, rendering it catalytically inefficient. Since the opening and closing motions govern the rate-determining product release, we conclude that optimal and coherent conformational fluctuations are necessary for efficient turnover of protein kinases.

Colocalization of fast and slow timescale dynamics in the allosteric signaling protein CheY.

It is now widely recognized that dynamics are important to consider for understanding allosteric protein function. However, dynamics occur over a wide range of timescales, and how these different motions relate to one another is not well understood. Here, we report an NMR relaxation study of dynamics over multiple timescales at both backbone and side-chain sites upon an allosteric response to phosphorylation. The response regulator, Escherichia coli CheY, allosterically responds to phosphorylation with a change in dynamics on both the microsecond-to-millisecond (µs-ms) timescale and the picosecond-to-nanosecond (ps-ns) timescale. We observe an apparent decrease and redistribution of µs-ms dynamics upon phosphorylation (and accompanying Mg(2+) saturation) of CheY. Additionally, methyl groups with the largest changes in ps-ns dynamics localize to the regions of conformational change measured by µs-ms dynamics. The limited spread of changes in ps-ns dynamics suggests a distinct relationship between motions on the µs-ms and ps-ns timescales in CheY. The allosteric mechanism utilized by CheY highlights the diversity of roles dynamics play in protein function.

Segmental motions, not a two-state concerted switch, underlie allostery in CheY.

The switch between an inactive and active conformation is an important transition for signaling proteins, yet the mechanisms underlying such switches are not clearly understood. Escherichia coli CheY, a response regulator protein from the two-component signal transduction system that regulates bacterial chemotaxis, is an ideal protein for the study of allosteric mechanisms. By using 15N CPMG relaxation dispersion experiments, we monitored the inherent dynamic switching of unphosphorylated CheY. We show that CheY does not undergo a two-state concerted switch between the inactive and active conformations. Interestingly, partial saturation of Mg2+ enhances the intrinsic allosteric motions. Taken together with chemical shift perturbations, these data indicate that the µs-ms timescale motions underlying CheY allostery are segmental in nature. We propose an expanded allosteric network of residues, including W58, that undergo asynchronous, local switching between inactive and active-like conformations as the primary basis for the allosteric mechanism.